In tumor microenvironment,interactions among multiple cell types are critical for cancer progression.To understand the molecular mechanisms of these complex interplays,the secreted protein analysis between malignant cancer cells and the surrounding nonmalignant stroma is a good viewpoint to investigate cell-cell interactions.Here,we develop two methods termed spike-in SILAC and triple-SILAC to quantify the changes of secretome in a co-cultured system of CT26 and Ana-1 cells(Figure 1).As results,compared with mono-cultured cells,188 and 56 secreted proteins were identified to change in the co-cultured system by spike-in SILAC and triple-SILAC methods respectively.The Figure 2 was shown three representative both-identified proteins(Galectin-1,Cathepsin L1 and Thrombospondin-1)by SILAC-based mass spectra(MS),in which pairs of isotope labeling peaks reflected the expression change of a protein in the co-cultured cell system.To validate the accuracy of MS analysis,cell supernatant was collected to detect the secretion level of the three proteins by Western blotting,and the results matched very well(Figure 3).So far the spike-in SILAC and triple-SILAC based quantitative MS approaches are sensitive to measure protein secretion changes due to cell-cell interactions of two different types of cells in vitro.